Detection and quantification of proteins in various biological samples is one of the most important and most often utilized assays with applications in research, medical diagnosis, early disease detection and detection of biological threat agents. While many protein detection methodologies are available (ELISA being a gold standard of those), they each have some limitations. The most common limitation is the time required to perform the assay and the complexity of the sample manipulation involved in the assay. Thus, there is a clear need for developing new rapid and homogenous protein detection methodologies. In Phase I of this project we established feasibility of a novel antibody-based homogenous protein detection methodology (molecular pincers) which allows detection of the presence of a target protein by a simple fluorescence intensity measurement. The assay is rapid and is technically extremely simple to perform (involves essentially no sample manipulation other then mixing the sample with the detection reagent). These characteristics make this new assay format particularly useful in situations where quick determination of the target protein is essential without a support of a laboratory equipped with sophisticated instrumentation (as in the case, for example, of point-of-care diagnostic assay). The long-term goal of this project will be to utilize molecular pincers to develop simple, rapid and inexpensive point-of-care diagnostic tests for a panel of cardiac disease markers. The goal of this Phase II project will be to develop molecular pincer assays for six markers of cardiac disease (troponin I, B-type natriuretic peptide (BNP), myoglobin, creatine kinase (CK-MB), C-reactive protein (CRP), and D-dimer) for research market. Completion of this goal will allow initiation of the process of approval of these assays for clinical diagnosis. Additionally, we will work on enhancing molecular pincer assay by developing strategies to improve assay sensitivity. Completion of this goal will enhance the commercialization prospects of molecular pincers by broadening the range of possible targets for which the assay could be developed. PUBLIC HEALTH RELEVANCE: Molecular pincer assays will find wide applications in research and diagnosis of human disease. In research, it could become more convenient, easier to use, less expensive replacement for currently used assays for detecting proteins. In diagnosis, it will allow more rapid and more straightforward detection of antigens correlated with human disease.